1. Field of the Invention
The invention is in the field of medicinal chemistry. In particular, the invention relates to certain antisense oligonucleotides and the use thereof for the treatment of cancer.
2. Background of the Related Art
Folate vitamins are essential components of intracellular metabolic pathways that transfer single carbon groups during nucleic acid and amino acid synthesis. In particular, folate is essential for de novo synthesis of deoxythymidylate triphosphate (dTTP), which incorporates into cellular DNA during DNA replication and repair.
Folate exists in three forms differing by oxidation state. Folic acid is the most oxidized form, whereas dihydrofolate and tetrahydrofolate are progressively more reduced. The tetrahydrofolate form recieves single carbon atom groups and transfers them in various biosynthetic pathways. During transfer, tetrahydrofolate is oxidized to dihydrofolate. Dihydrofolate is in turn reduced by the enzyme dihydrofolate reductase (DHFR), completing a metabolic cycle and preparing tetrahydrofolate to again receive single carbon atom groups.
Folate inhibition impairs cell growth, and thus folate-dependent metabolic pathways have long been exploited as chemotherapeutic targets. For example, the folate analog methotrexate (MT) is a successful antifolate chemotherapy agent due to its potent inhibition of DHFR. Newer antifolates are now in clinical trials.
Folates and antifolates enter cells via two major transport systems. The predominant carrier in normal tissue has a high affinity for reduced folates, and relatively low affinity for folic acid. These biochemical characteristics account for its name, reduced folate carrier (RFC). The second transport system includes transmembrane folate receptor (FR) isoforms, referred to as FR-α, FR-β, and FR-γ. These receptors have high affinity for folic acid and intermediate affinity for reduced folate forms.
In most normal cells, there is little or no expression of FR isoforms, and their physiologic role, if any, remains to be determined. Interestingly, however, FR isoforms are known to be expressed in various malignant tissues. In particular, FRα is highly expressed in many carcinomas of epithelial cell origin, such as ovarian carcinoma. It is possible that FR expression is related in some way to the abnormal growth characteristics of such cells.
Antifolates such as MTX are transported avidly by the RFC, but not by FR isoforms. In cancer cells, reduced RFC expression may promote chemotherapy resistance by reducing the cell's ability to take up antifolates. In such cancer cells, the FR isoforms may serve as an alternative folate transport mechanism, thereby promoting cancer cell growth.
In early 1990s, Matsue et al., (Proc. Natl. Acad. Sci. U.S.A. 89: 6006-6009, 1992) and Luhrs et al. (J. Clin. Invest. 90: 840–847, 1992) observed that expression of FRa enabled cultured mouse keratinocyte and fibroblasts cells to grow in low folate media. Matsue et al. suggested that cells vary in their folate requirement, with rapidly proliferating cells such as malignant cells requiring high levels of folate to survive. They further intimated that the growth potential of malignant cells may be directly related to the level of folate receptor expression.
About the same time, in 1991, Westerhof et al. (Cancer Research 51: 5507–5513, 1991) reported on experimental results that concluded that FRa expression enhanced antifolate uptake in L1210 cells. In addition, they showed that FRa mediated uptake of thymidylate synthase inhibitors, a related class of anticancer agents. Importantly, their data demonstrated that FRα expression markedly inhibited the growth of L1210 cells when the cells were exposed to antifolates or thymidylate synthase inhibitors. Hence, began a line of research focusing on the increased expression of FRα in malignant cells and its use for specific targeting and delivery of anti-cancer drugs to malignant cells.
In 1993, Chung et al. (J. Clin. Invest. 91: 1289–1294, 1993) published the results of their investigation on FRα-mediated transport in a methotrexate resistant human breast cancer cell line (MCF-7 cells). The investigators stably transfected MCF-7 cells with human FRα cDNA, and evaluated cell growth, and folate and antifolate transport. Although, there was a positive correlation between cell growth and FRα expression when cells were grown in low folate media, they observed that FRα expression also enhanced MTX uptake and cellular sensitivity to the negative growth effects of MTX. Hence, in line with the findings of Westerhof et al., it was suggested that the increased expression of FRα would be desirable as it provided a means for cancer therapy using the chemotherapeutic drug, MTX.
Subsequently, Sun et al. (J. Clin. Invest. 96: 1535–1547, 1995) transduced human cervical carcinoma (HeLa) cells with recombinant adeno-associated viruses (AAV) expressing FRα in either sense or antisense direction. As expected, the antisense construct suppressed FRα expression, whereas the sense construct increased FRα expression. When cells were grown in supraphysiological levels of folate, suppression of FRα expression with antisense had little effect on cellular proliferation, similar to the control (untransduced cells), and FRα sense expression reduced cellular proliferation. Similar results were obtained in vivo: when transduced cells were transferred to nude mice, cells transduced with antisense FRα grew larger tumors than untransduced cells or sense FRα-transduced cells. The investigators concluded that “there was an inverse relationship between FR expression and cell proliferation in the cells” (emphasis added).
In the same year, Spinella et al. (J. Biol. Chem. 270: 7842–7849, 1995) reported having stably transfected L1210 human leukemia cells with a cDNA expressing FRα. L1210 cells express a nonfunctional reduced folate carrier, and are MTX-resistant. It was observed that FRα-transfected cells were capable of taking up MTX, thereby tending to restore antifolate sensitivity to the cells. Hence, their results seemed to confirm that the high expression of FRα would be desired as it seemed to enhance sensitivity to chemotherapeutic agents.
Gorlick et al., in their 1996 publication (New England Journal of Medicine 335: 1041–1048, 1996), reviewed the problem of methotrexate resistance in acute leukemias. These authors concluded that and related receptors had little or no role in leukemia therapy or antifolate resistance. Instead, they suggested targeting other points in the folate metabolic pathway, such as the dihydrofolate reductase gene.
In 1999, also consistent with the developing line of investigation, Sun et al. (Cancer Research 59: 940–946, 1999) published their results for their research on signal transduction in FRα-transduced HeLa cells, using a protocol essentially identical to that described in Sun et al., 1995. They observed that FRα expression directly correlated with increased thymidine kinase activity in sense and antisense FRα-transduced HeLa cells. Increased thymidine kinase activity induced by FRα increased sensitivity of HeLa cells to azathymidine (AZT), another anticancer chemotherapeutic agent. Antisense suppression of FRα expression reduced thymidine kinase activity and reduced sensitivity to AZT, yet, another indication that antisense supression of FRα was not desirable as it renderd the anti-cancer drug, AZT, ineffective against the disease.
The above references are incorporated by reference herein where appropriate for appropriate teachings of additional or alternative details, features and/or technical background.